探討高膽固醇飲食誘發發炎反應之病生理學、藥物治療與新穎影像技術之應用

Abstract

高膽固醇血症為動脈粥狀硬化性疾病的主要危險因子，對人類健康形成重大威脅。目前盛行的理論認為在高膽固醇血症中，於內皮下空間累積的低密度脂蛋白可造成動脈血管壁的過氧化壓力及慢性發炎，因此減弱動脈對物理性或化學性刺激的抵禦，促成粥狀動脈硬化的起始與進展。由於動脈不是唯一暴露於低密度脂蛋白的組織，本論文提出假說，認為高膽固醇血症在動脈以外亦可能引發發炎現象而導致組織或器官的功能失常。 基於此一假設，本論文首先探討高膽固醇血症在心肌引發的發炎現象。在第二章中我們以十二周高膽固醇飲食於紐西蘭白兔誘發極度高膽固醇血症，並利用序列性磁振造影探討心肌功能性變化。我們發現在此模式中高膽固醇血症並不造成冠狀動脈阻塞以及心肌灌流缺失，也不影響左心室質量與射血分率，但心肌微循環滲漏性則顯著提升。此現象肇因於冠狀動脈系的血管新生，使心肌微循環內微血管表面積與單位面積滲漏性增加。我們同時也發現高膽固醇血症伴隨著血漿中磷脂醯膽鹼過氧化物(PCOOH)及IL-8的濃度上升，以及心肌遭巨噬細胞浸潤。以上因素都可能與冠狀動脈系的血管新生有關。雖然高膽固醇血症在兔心肌可能引起發炎現象，但此動物模式中過高的總膽固醇使得其結論的臨床相關性降低。因此我們在第三章中以三周高膽固醇飲食在大鼠引發溫和的高膽固醇血症，並分析在此模式中心臟功能的變化。研究結果發現高膽固醇血症在心肌可增加過氧化壓力，並引起巨噬細胞浸潤。針對生理功能的分析發現在此動物模式中微循環滲透性不受高膽固醇血症影響；心肌固有收縮力雖然下降，但左心室收縮反應增強，心跳增快，同時交感神經系統的活性亦顯著上升。以apocynin與ICAM-1單株抗體投與此等大鼠，心肌發炎與力學功能的交感神經過度活性可獲得改善。由以上證據，我們推測高膽固醇血症會經由過氧化壓力與發炎反應抑制心肌的固有收縮力，交感神經系統因而活化以為代償。有鑒於自由基與過氧化壓力在心臟病生理學的重要性，我們在第四章提出一套新穎技術，以dihydroethidium作為氧屬自由基的螢光探針，將高速螢光攝影術應用於離體灌流心臟，在活組織中記錄自由基隨生理/病理性刺激的改變。此系統可成功地偵測缺血再灌流過程中心肌內氧屬自由基的時序性變化。我們期待改進效能後此系統能應用於研究高膽固醇血症對心肌自由基動態的影響。 本論文的第五章著墨於高膽固醇血症在肝臟引發的發炎現象。肝臟是體內主要負責膽固醇代謝的器官，其生理配置使肝臟能負荷大量的膽固醇流量而不致引起細胞傷害，但高膽固醇血症是否可在肝臟誘發發炎則仍屬未知。以三周高膽固醇飲食誘發大鼠之高膽固醇血症後，我們發現血漿肝功能指數上升、門脈壓升高、肝組織纖維化與肝脾腫大。於組織切片中可發現肝實質中呈巨噬細胞浸潤並伴隨輕度門脈旁纖維化，進一步以二諧頻顯微術檢視活肝組織可發現其中的膠原蛋白結構在高膽固醇血症影響下出現顯著改變。肝組織之脂質分析顯示膽固醇主要以酯化型態累積於肝細胞中。利用活體雙光子顯微術測量活體動物中肝細胞自體螢光強度，我們發現高膽固醇血症大鼠的肝細胞中過氧化壓力呈顯著增加，同時此現象亦可由分析肝組織中TBARS與glutathione等過氧化壓力的生化指標得到證實。由於高膽固醇血症造成肝組織中gp91phox的表現增加，而且投與apocynin可逆轉高膽固醇血症引起的過氧化壓力及肝炎現象，故我們認為NADPH oxidase在高膽固醇血症造成脂肪性肝炎的病生理學中扮演重要角色。 本論文的第六章針對高膽固醇血症與血液中單核球活化的關係進行探討。由於前述研究發現高膽固醇血症引起心肌與肝臟中的巨噬細胞浸潤現象，而周邊循環中的單核球是組織發炎反應中巨噬細胞的主要來源，因此周邊循環單核球的活化可能是高膽固醇血症造成組織發炎的重要機轉。我們提出假說，認為在高膽固醇血症中過量的原型低密度脂蛋白在周邊循環中可直接活化單核球，引發過氧化壓力並促進單核球與內皮細胞的黏附，誘發微循環發炎現象。為驗證此假說，我們分離正常大鼠全血中的白血球，以原型與氧化型低密度脂蛋白刺激後觀察單核球細胞內過氧化壓力與表面黏附因子CD18之表現。研究發現原型低密度脂蛋白較氧化型低密度脂蛋白更能有效增加單核球細胞內氧化壓力，並發現細胞內鈣離子增加、肌動蛋白聚合以及NADPH oxidase活化均參與其訊息傳遞過程。同時原型低密度脂蛋白亦可增加單核球表面CD18的表現。此等現象可重現於高膽固醇大鼠體內，並且細胞內過氧化壓力以及過量的表面黏附因子促使白血球於提睪肌微循環中滾動、黏附與穿越，造成微循環發炎。Rosiglitazone除了用於治療第二型糖尿病，近年來亦發現具有強大抗發炎活性，故本實驗利用此發炎模式探討rosiglitazone的效果。我們發現rosiglitazone可透過影響肌動蛋白之聚合與NADPH oxidase的活性，降低原型低密度脂蛋白造成的單核球細胞內過氧化壓力。同時在高膽固醇大鼠口服投予rosiglitazone亦可改善高膽固醇血症引發的單核球活化與微循環發炎現象。以上結果顯示rosiglitazone可能對改善高膽固醇血症造成的微循環發炎性病變具有療效。 綜上所言，本論文發現低密度脂蛋白不需氧化修飾亦可活化周邊單核球，進而誘發提睪肌微循環的發炎反應，並利用兩種高膽固醇血症的動物模式，證明高膽固醇血症誘發的發炎反應不只侷限於動脈血管壁，亦可及於心肌及肝組織之微循環。這些研究顯示NADPH oxidase衍生的過氧化壓力在此機制中是重要的致病機轉，也有希望成為藥物治療的新穎標靶。慢性微循環發炎可能影響正常組織功能，並釋放促發炎因子，促進動脈硬化的進展。我們期待更多的轉譯性研究以釐清微循環發炎的病生理學以及臨床相關性。

Hypercholesterolemia (HC) is a major risk factor for atherosclerotic vascular diseases. Current theory emphasizes the role of low-density lipoprotein (LDL) since its subintimal accumulation results in oxidative stress and inflammation of the arterial wall, which predispose the vessel to atherogenesis upon various physical/chemical noxious stimuli. Considering the artery is not the only tissue exposed to excess LDL during HC, we propose a hypothesis that HC would induce tissue inflammation and disturb physiologic function outside the arterial wall. We first targeted the heart to investigate this hypothesis with a rabbit model of HC. In this model, extreme HC was induced in New Zealand white rabbits by 12 weeks of 0.5% cholesterol-enriched diet. Left ventricular function and microcirculatory characteristics were determined with serial cardiac magnetic resonance imaging. We found that HC significantly enhanced microvascular leakage while regional perfusion, left ventricular mass, and ejection fraction were comparable to the control group. Morphologically there were increased macrophage infiltration and coronary neovascularization in the myocardium. Parallel increases in serum levels of phosphatidylcholine hydroperoxide (PCOOH) and IL-8 might provide a link between myocardial inflammation and angiogenesis. We also examined the effects of mild HC on cardiac function in a rat model. In this model, the rats were fed 4% cholesterol-enriched diet for 3 weeks, and oxidative stress as well as macrophage accumulation in the heart led to a totally different phenotype. While Evans blue leakage remained unchanged and intrinsic contractility was depressed, HC induced chronotropism, inotropism, and sympathetic hyperactivity of the rat heart. These phenomena were improved by systemic administration of either apocynin or intercellular adhesion molecule-1 (ICAM-1) neutralizing antibody. We concluded that oxidative stress and inflammation might depress contractile function in HC hearts, and as a result sympathetic activation ensues to maintain functional homeostasis. As it is important to understand the role of free radicals in cardiac pathophysiology, we developed an optical mapping system to detect reactive oxygen species in isolated perfused heart preparation. The system successfully recorded the dynamics of reactive oxygen species throughout global ischemia reperfusion. Adequate modification is necessary to apply the system in the research of free radicals in HC hearts. We next investigated HC-induced steatohepatitis. The liver experiences large flux of cholesterol everyday, but it is spared from cholesterol toxicity owing to effective esterification machinery and a tight control of cholesterol uptake. It is not clear, however, whether or not HC would overwhelm cholesterol-handling capability of the liver and induce steatohepatitis. In cholesterol-fed rats as mentioned above, the liver accumulated cholesteryl ester as well as triglycerides. We found elevated serum activities of alanine aminotransferase (ALT), aspartate transaminase (AST) and alkaline phosphatase (Alk-p), enhanced Evans blue extravasation, as well as increased macrophage infiltration; all indicated the presence of steatohepatitis. In addition we also found hepatosplenomegaly, portal hypertention and liver fibrosis in HC liver. Biochemical parameters such as tissue thiobarbituric acid-reactive substances (TBARS), glutathione (GSH) and GSSG (glutathione disulfide) as well as in vivo NADPH-derived autofluorescence indicated an increased oxidative burden in hepatocytes of HC rats. Since gp91phox was upregulated in HC livers and coadministration of apocynin effectively treated hepatic inflammation, NADPH oxidase must play a role in the pathogenesis of HC-induced steatohepatitis. In previous studies macrophages activation and infiltration were noted in the heart and the liver during HC. Since monocytes are the major source of inflammatory macrophages, we hypothesized that direct activation of monocytes by LDL in peripheral circulation contributes to HC-induced microvascular inflammation. We first demonstrated that both native- and copper oxidized- LDL elicited intracellular respiratory bursts in circulating monocytes in a time- and dose-dependent manner. In this experiment we found native LDL was a stronger stimulus, and subsequent studies showed that intracellular calcium mobilization, actin polymerization and NADPH oxidase activation were involved in the signaling pathway. Similar phenomena were observed in monocytes from HC rats, and augmented cellular oxidative stress as well as surface CD18 abundance resulted in increased leukocytes-endothelial interaction in cremaster microcirculation. Rosiglitazone as an insulin sensitizer and a potent anti-inflammatory agent was effective in treating HC-induced microvascular inflammation. It did so partly by reducing actin polymerization and NADPH oxidase activity in peripheral monocytes. To sum up, we provided experimental evidences in the heart and the liver to support our hypothesis that HC could induce inflammation and organ dysfunction beyond arterial atherosclerosis. We also proposed that native LDL activation of peripheral monocytes as one of the general pathogenic mechanism. As NADPH oxidase interwove these phenomena, it possibly will be an important target to develop novel therapeutic strategies. The clinical relevance of chronic microvascular inflammation remained obscure, and we are looking forwards to well-designed translational studies to unravel its significance.